Human granulocytic ehrlichiosis (HGE) is an emerging disease of humans caused by a novel, uncharacterized ehrlichial agent. In 1994, this severe, potentially fatal, tick-borne infection was identified in North American areas coincident with regions endemic for Lyme disease and babesiosis. Although rarely diagnosed, the fatality rate approaches 10% of cases and seroprevalence rats are high in some areas. The taxonomic positions of this agent and Ehrlichia equi, the causative agent of equine granulocytic ehrlichiosis (EGE), have been difficult ot establish because of the inability to cultivate these organisms in vitro. The agent of HGE has been isolated by infection of horses with human blood, and may now be studied more directly. The long term goals of this project are to characterize the etiologic agent and pathogenesis of HGE in order to develop strategies for diagnosing, preventing, and treating this emerging infection. The primary hypothesis is that the agent of HGE is distinct from other agents of human ehrlichiosis, yet is similar or identical to E. equi. Thus, the comparative ecology and pathogenesis of EGE relates directly to HGE and human health. Specific aims for t e sting this hypothesis are; 1. to determine the major immunoreactive, surface-exposed proteins of the agent of HGE. Purified HGE agent will be used in immunoblots with anti-ehrlichia sera a nd monoclonal antibodies. HGE agent cell-surface antigens will be demonstrated by immuno-electron microscopy and extrinsic labeling. 2. to clone and sequence two novel genes for immunodominant, surface- exposed proteins of the HGE agents. Genes encoding immunodominant, surface-exposed proteins of HGE will be cloned from a genomic library and sequenced. The sequences will be compared with homologs in E. equi and other ehrlichiae. 3. to determine the tissue and cell tropism of the HGE agent in the horse model and in cell culture. Infected horses will be examined to identify infected tissues and cell types using PCR, immunohistology, and flow cytometry. Co-cultivation of persistently infected equine bone marrow with primary mammalian cells, cell lines, and tick cell lines will be used to establish the in vitro biological tropism. 4. to demonstrate the unique HGE biology by virtue of the specificity of the interactions of rickettsiae, tick vectors, and hosts in transmission via Ixodes scapularis ticks. Ixodes scapularis ticks of all stages will feed on an infect ed horse, and each subsequent stage will be tested for the ability to transmit the infection to a susceptible horse. Tick tissues will be examined for the presence and distribution of the HGE agent by PCR, immunohistology, and ultrastructure. In addition to establishing tahe identify of the HGE agent, important derivatives of these experiments will be the determination of the mechanism of transmission of the HGE agent and elucidation of early steps in pathogenesis of infection at athe tissue and cellular level. Such experiments will speed identification of: i) the natural reservoir, ii) the mechanism of spread of the infection; iii) the mechanism of natural maintenance; and, iv) the mechanisms by which humans contract and develop HGE. All of these are imperative to assess the degree of human risk and in developing strategies for control and prevention of this emerging infection